Chromatin immunoprecipitation (ChIP) is an important technique used in the study of DNA/protein interactions. An advantage of ChIP is that it can be used for analysing the association of specific proteins, or their modified isoforms, with defined genomic regions. A review of existing ChIP technology is provided in O'Neill et al. (2003) “Immunoprecipitation of native chromatin, NChIP”, Methods: A Companion to Methods in Enzymology 31:76-82. ChIP may be used to determine whether proteins such as transcription factors and modified histones bind to a particular region on the endogenous chromatin of living cells or tissues.
In a ChIP assay, fragments of the DNA-protein complex (i.e. the chromatin) are prepared in such a way so as to retain the specific DNA-protein interactions. These chromatin fragments can then be immunoprecipitated using an antibody against the protein present in the complex. The isolated chromatin fraction can then be treated to separate the DNA and protein components. The identity of DNA fragments isolated in connection with a particular protein (i.e. the protein against which the antibody used for immunoprecipitation was directed) can then be determined by Polymerase Chain Reaction (PCR), Real Time PCR (qPCR), hybridization on microarrays, direct sequencing or other technologies used for identification of DNA fragments of defined sequence.
Hence, a chromatin immunoprecipitation assay typically involves the following five key steps: (i) preparation of chromatin to be analysed from cells; (ii) immunoprecipitation of chromatin using an antibody; (iii) isolation of the precipitated chromatin fragments; (iv) DNA recovery from the precipitated product; and (v) DNA analysis.
The ChIP technique has two major variants that differ primarily in how the starting (input) chromatin is prepared. The first variant (designated NChIP) uses native chromatin prepared by micrococcal nuclease digestion of cell nuclei by standard procedures. The second variant (designated XChIP) uses chromatin cross-linked by addition of formaldehyde to growing cells, prior to fragmentation of chromatin, usually by sonication. Some workers have used mild formaldehyde cross-linking followed by nuclease digestion, and UV irradiation has been successfully employed as an alternative cross-linking technique.
Typically the immunoprecipitation of chromatin fragments is performed using an antibody specific to the protein of interest which is bound to DNA. The antibody-bound chromatin fragments may be isolated from the sample using a solid phase.
WO 2012/076882 describes a separation column comprising a chamber for holding a liquid sample comprising chromatin, and a rigid porous matrix on which a ligand is immobilized, wherein the ligand is capable of binding to a protein associated with the chromatin. In use, the liquid sample may first be added to a chamber in a separation column, e.g. through an upper opening in the column. The liquid sample may then pass through a rigid porous matrix, typically positioned above an effluent port at a lower end of the column, and thereby exit the column. In this way, chromatin fragments present in the liquid sample can bind to the ligand whilst passing through the matrix. Chromatin fragments are thereby separated from the liquid sample, which may then be discarded.
However, when the apparatus described in WO 2012/076882 is used, especially in a spin column, in order to obtain the necessary contact between the chromatin and the ligand on the rigid porous matrix to secure its retention to the matrix, it is necessary for the liquid sample containing the chromatin to be added in a volume such that it is completely absorbed by the matrix, i.e. the liquid sample must be retained within the internal void space of the matrix. Any volume of the liquid sample which exceeds the void volume of the matrix will not be able to contact the functional groups on the ligand.
At the publication date of WO 2012/076882, it was thought the apparatus could operate on a liquid sample roughly equal to the internal void volume of the rigid porous matrix. However, subsequent to the publication of WO 2012/076882, the present inventors have found that, in practice, when used in a spin column, the amount of liquid sample is restricted to around 2-3 times the void volume of the rigid porous matrix to ensure adequate chromatin recovery. For example, in a standard spin column where the void volume is about 40 μl, the apparatus is typically restricted to chromatin-containing liquid sample volumes of a maximum of 100 μl.
A further drawback of the apparatus described in WO 2012/076882 is that it is generally necessary in practice to draw the liquid sample through the matrix by centrifugation: multiple centrifugation processes are typically required. This frequently requires complex and expensive machinery, such as robot arms, to carry out the method, particularly when used for assays involving multi-well plates.
Pipettes are laboratory tools commonly used in a wide variety of scientific fields to transport a measured volume of liquid, often as a media dispenser. Typically, pipettes work by creating a partial vacuum above the liquid-holding chamber such that reduced pressure causes the liquid sample to be drawn into the pipette and selectively releasing this vacuum (i.e. increasing the pressure) to expulse the liquid.
US 2008/0119637 describes a pipette tip column comprised of a packed bed of gel resin, wherein the packed bed of gel resin is comprised of agarose or sepharose, and wherein the gel resin is further comprised of an affinity group having an affinity for the protein analyte, and wherein said gel resin lacks residual ion exchange groups. The agarose gel is held between two frits in the tip. The protein analyte may be extracted by passing a sample solution through the pipette tip column. Agarose gels are prone to non-specific binding of DNA and proteins, and it is difficult to provide adequate washing steps to reduce the resulting background signal. These disadvantages are also encountered when the agarose gel forms part of an extraction system in a pipette tip.
US 2010/0009845 describes a pipette tip which is fitted with a porous organic monolith which is doped with active particles. The tip can be used as a tool for solid phase extraction, especially for desalting, isolating and purifying biomolecules such as peptides and proteins. According to the process described in this document, polymerisation may take place in situ, so that the product must be custom made for each application or has to be made by the user. Using this process, it would also be difficult to achieve reproducibility between the monoliths.
The application of reduced pressure to draw in a liquid sample and increasing the pressure to expulse the liquid can avoid the need for centrifugation. However, when this process is carried out in a standard size separation column with a standard sized frit of the type described generally in WO 2012/076882, driving the liquid through the column using reduced or increased pressure can cause foaming. This foaming can cause contamination or loss of sample and can block further liquid passage.
Pipette tips having filtration apparatus, such as frits disposed therein are generally known in the art. However, in the known pipette tips, the filtration apparatus is typically positioned in the top half of the tip, close to the point where the tip engages the main body of the pipette. The function of such apparatus is generally to prevent liquid from entering the aspiration means for drawing the liquid in and protect this equipment, rather than to capture an analyte such as chromatin present in the liquid.
Some known pipette tips such as the ZipTip® manufactured by Merck Millipore have filtration or analyte capture means disposed in the lower half of the tip. However, these generally comprise fibrous material with general sorbents such as C8 or C18 modified silica. Pipette tips containing chromatin-specific capture matrices have not previously been disclosed in the art.
Thus there is a need for improved chromatin immunoprecipitation assay apparatus and methods which address one or more of the above problems.